Click chemistry–enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling

2021 ◽  
Vol 118 (48) ◽  
pp. e2025265118
Author(s):  
Timothy W. Bumpus ◽  
Shiying Huang ◽  
Reika Tei ◽  
Jeremy M. Baskin
Keyword(s):  
Mammalian Cells ◽  
Regulatory Networks ◽  
Glycogen Synthase ◽  
Second Messengers ◽  
Fluorescent Labeling ◽  
Glycogen Synthase Kinase 3 ◽  
Kinase C ◽  
Genome Wide ◽  
Phenotype Selection ◽  
Fluorescent Tagging

Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.

scholarly journals Convergence of Multiple Signaling Cascades at Glycogen Synthase Kinase 3: Edg Receptor-Mediated Phosphorylation and Inactivation by Lysophosphatidic Acid through a Protein Kinase C-Dependent Intracellular Pathway

2002 ◽  
Vol 22 (7) ◽  
pp. 2099-2110 ◽  
Author(s):  
Xianjun Fang ◽  
Shuangxing Yu ◽  
Janos L. Tanyi ◽  
Yiling Lu ◽  
James R. Woodgett ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Lysophosphatidic Acid ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Hek293 Cells ◽  
Glycogen Synthase Kinase 3 ◽  
Kinase C ◽  
Pkc Inhibitors

ABSTRACT Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor β subunit (PDGFRβ) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRβ mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cγ (PLCγ) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRβ. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCγ-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.

scholarly journals GSK-3β activation is required for ZIP-induced disruption of learned fear

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sukwoon Song ◽  
Jihye Kim ◽  
Kyungjoon Park ◽  
Junghwa Lee ◽  
Sewon Park ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Glycogen Synthase ◽  
Conditioned Fear ◽  
Memory Loss ◽  
Glycogen Synthase Kinase 3 ◽  
Inhibitory Peptide ◽  
Kinase C ◽  
Learned Fear ◽  
Gsk 3Β

Abstract The myristoylated zeta inhibitory peptide (ZIP), which was originally developed as a protein kinase C/Mζ (PKCζ/PKMζ) inhibitor, is known to produce the loss of different forms of memories. However, ZIP induces memory loss even in the absence of PKMζ, and its mechanism of action, therefore, remains elusive. Here, through a kinome-wide screen, we found that glycogen synthase kinase 3 beta (GSK-3β) was robustly activated by ZIP in vitro. ZIP induced depotentiation (a cellular substrate of memory erasure) of conditioning-induced potentiation at LA synapses, and the ZIP-induced depotentiation was prevented by a GSK-3β inhibitor, 6-bromoindirubin-3-acetoxime (BIO-acetoxime). Consistently, GSK-3β inhibition by BIO-acetoxime infusion or GSK-3β knockdown by GSK-3β shRNA in the LA attenuated ZIP-induced disruption of learned fear. Furthermore, conditioned fear was decreased by expression of a non-inhibitable form of GSK-3β in the LA. Our findings suggest that GSK-3β activation is a critical step for ZIP-induced disruption of memory.

scholarly journals Lithium Chloride Sensitivity Connects the Activity of PEX11 and RIM20 to PGM2 translation

2022 ◽  
Author(s):  
Ashkan Golshani ◽  
Sasi Kumar Jagadeesan ◽  
Mustafa Algafari ◽  
Maryam Hajikarimlou ◽  
Sarah Takallou ◽  
...  
Keyword(s):  
Lithium Chloride ◽  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Cell Signalling ◽  
Sugar Metabolism ◽  
Galactose Metabolism ◽  
Gene Encoding ◽  
Kinase C ◽  
Downstream Effects ◽  
Signalling Transduction

Abstract Lithium chloride (LiCl) is a widely used and extensively researched drug for the treatment of bipolar disorder (BD). As a result, LiCl has been the subject of research studying its toxicity, mode of action, and downstream cellular responses. LiCl has been shown to influence cell signalling and signalling transduction pathways through protein kinase C and glycogen synthase kinase-3 in mammalian cells. LiCl's significant downstream effects on the translational pathway necessitate further investigation. In yeast, LiCl is found to lower the activity and alter the expression of PGM2, a gene encoding a sugar-metabolism phosphoglucomutase. When phosphoglucomutase activity is reduced in the presence of galactose, intermediates of galactose metabolism aggregate, causing cell sensitivity to LiCl. In this study, we identified that deleting the genes PEX11 and RIM20 increases yeast LiCl sensitivity. We further show that PEX11 and RIM20 regulate the expression of PGM2 mRNA at the translation level. The observed alteration of translation seems to target the structured 5′-untranslated region (5′-UTR) of the PGM2 mRNA.

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